Chromatographic process utilizing a fluidized bed

ABSTRACT

A liquid chromatographic process performed at least partly in a fluidised bed contained in a vessel and comprising particle fluidised by an upwardly directed liquid flow, said process comprising (a) a capture step in which one or more compounds of a sample are captured by the particles and (b) a wash and/or releasing step in which the particles is in form of a fluidised bed through which a liquid flow is passing. The characteristic feature is that the liquid (liquid 1) used in the wash or the releasing step (step 1) is immediately followed by a liquid (liquid 2, step 2) having a higher density than liquid 1 while maintaining the bed in a fluidised state. A liquid chromatographic process having an actual sequence of steps comprising (c) at least a capture step in which a sample deriving from an animal is bound to the particles and (d) two consecutive steps (step 1 and step 2) in which the bed is fluidised by a liquid flow passing through said vessel. The characteristic feature of the process is that the liquid used in step 2 (liquid 2) has a density that is higher than the density of the liquid used in step 1 (liquid 1).

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/SE99/01965 filed Oct. 31, 1999.

TECHNICAL FIELD

The present invention concerns a new process for performing liquidchromatography in which there is a sequence of steps of which at leasttwo consecutive steps (step 1 and step 2) are in fluidised bed mode bythe use of an upward flow.

With respect to various modes of the invention reference is made tocopending International Patent Application derived from SE 9803813-6 andSE 9803737-7. This International Patent Application is herebyincorporated by reference.

BACK GROUND TECHNOLOGY

Liquid chromatographic processes are carried out on particle matrices inform of packed or fluidised beds. The processes typically contain atleast one step according to type (b) below and one or more functionalsteps selected from the remaining types of steps (a,c,d,e,f):

a) equilibrating the particles with a liquid conditioning the particlesfor capture/binding;

b) capturing one or more compounds present in a liquid sample by theparticles;

c) washing the particles to which said one or more compounds have becomebound;

d) releasing at least one of said one or more compounds from theparticles;

e) cleaning the particles; and

f) regenerating the particles.

The capture step (type b) together with the selected steps define anactual sequence in a particular chromatographic process. In an actualsequence there may also be steps other than those outlined above (a-f).A typical sequence comprise the sequence

a,b,c,d,e,f(a),b,c,d,e,f(a) . . . possibly with extra steps inserted inthe sequence given. f(a) means that step a and step f may coincide andthat chromatographic processes can be cyclic.

In each step the particles are treated with an appropriate liquid(solution/buffer) that is aqueous or non-aqueous.

The term “capture” includes that the compound becomes bound to theparticles. The binding may occur via the formation of affinity bonds,covalent bonds, entrapment within the particles etc. Examples ofaffinity are bioaffinity, ionic interaction, hydrophobic interactionetc. The captured compound may be a compound that is to be purified or acontaminant that one wants to separate from another compound or removefrom the liquid used in the capture step.

The liquid used in the releasing step typically contains an agent thatwill release the captured compound, for instance a buffer giving anappropriate pH, a salt giving an appropriate ionic strength, a substancethat competitively will inhibit the binding between the capturedcompound and an affinity ligand/structure on the particles, etc. Theterm “release” includes release through breaking of affinity bonds,covalent bonds etc. Covalent bonds can be broken by chemical reactionsor enzymatically.

The liquid used in a step can change continuously or step wise during astep. Releasing by the aid of a gradient, for instance, is typical forelution on packed beds but has been rare on fluidised beds (Shiloach etal., Sep. Sci. Techn. 34(1) (1999) 29-40). Another example is changing awashing solution during a washing step.

In packed beds, the releasing step typically can consist of one or moresubsteps. For instance the capture step may mean capture of two or morecompounds that bind differently to the particles. For release, thecompounds may require different conditions and different compositions ofthe liquid.

Steps can wholly or partly coincide. The regeneration step, forinstance, is primarily related to regeneration of the particles to beused in a second cycle of the process and then coincides with theequilibration step of the second run. The capture step can mean that thecompound is only retarded suggesting that the releasing step is at thesame time ongoing. In case a contaminant is captured by the particles,possibly in combination with passing through the compound to bepurified, release can take place in the cleaning step.

Cleaning steps are often called cip (=cleaning in place). Cip-stepsnormally comprise high concentration of solutes, such as NaOH, in theliquid used. This means that the liquid for cleaning often has thehighest density in an actual sequence.

Each step can be run in a fluidised or packed bed mode with verticalflow that either may be upward or downward. The flow direction mayswitch between different steps. Plug flow has often been of advantage inchromatography, in particular in capture steps.

The same or different vessels can be used for the various steps of anactual sequence.

During the various steps the particles are placed in a vessel as knownin the field. Se WO 9520427 (Amersham Pharmacia Biotech AB), WO 9218237(Amersham Pharmacia Biotech AB), our copending International PatentApplication derived from SE 9803813-6 and SE 9803737-7 etc. Suitablevessels have an inlet end and an outlet end. The vessel is typicallyplaced vertically with the outlet pointing vertically upwards on the topside and the inlet pointing vertically downwards on the bottom side. Itcan also be the other way round. The inlet and outlet function,respectively, may comprise one or more openings into the vesselinterior.

BACK GROUND PUBLICATIONS

Density differences between liquids used in consecutive steps have beenused previously in model experiments of fluidised bed purification.These experiments have included various concentrations of glycerol inthe washing solution for small scale fluidised bed treatments. Thepurpose has been to increase the viscosity, and possibly also thedensity, of the washing solution compared to the solution applied forthe adsorption/capture step. See Draeger & Chase, Bioseparation 2 (1991)67-80; Chase et al, Sep. Sci. Techn. 27 (1992) 2021-2039; Chase et al,J. Chromatog. 597 (1992) 129-145; Chase et al, 6^(th) European Congressof Biotechnology (ECB 6), Florence, Italy, Jun. 13-17, 1993; Chase,TIBTECH 12 (1994) 296-303; Chang et al, Biotechn. Bioengin. 48 (1995)355-366; and Chang et al, Biotechn. Bioengin. 49 (1996) 204-216). Thearticles discuss that there are certain disadvantages on the subsequentelution (releasing) step due to the viscosity created by the addedglycerol and that these disadvantages can be avoided by running thesubsequent releasing step in a packed bed mode.

In fluidised bed chromatography liquids of increased densities haveoften been used when going from an equilibration step to a capture step(the samples are often is relatively dense).

Recently gradient elution has been applied in fluidised bedchromatography. See Shiloach et al., Sep. Sci. Techn. 34(1) (1999)29-40.

DRAWBACKS OF PREVIOUS TECHNIQUES

In the releasing step of an actual sequence defined above the liquidused contains an agent that will release a captured compound from theparticles. This means that the density of a liquid will tend to increaseduring the releasing step. In case the bed is fluidised by an upwardflow there will be a tendency that liquid containing the releasedcompound will be transported downwards simultaneously with the front ofthe release liquid progressing upwards. The result will be a dilution ofthe released compound and many times an unfavourable increase in thevolume of liquid to be handled in the subsequent processing of thereleased compound.

Washing liquids may be relatively light. If a washing liquid has adensity lower than the density of the liquid of a preceding step it willcause turbulence and lowered efficiency of the washing step. This isparticularly pronounced for washing steps that are consecutive toa-capture step because the liquid used in a capture step many times isrelatively dense.

These drawbacks will be more pronounced in vessels that do not have amovable outlet adapter compared to vessels that have this type ofadapter.

THE INVENTION

The invention is a method for processing a liquid sample containing oneor more compounds to be removed by capturing at to least one of them byparticles that are brought into contact with the sample. The methodcomprises an actual sequence as defined above comprising a part sequenceof at least two consecutive steps (step 1 and step 2) in which theparticles are fluidised. Step 1 precedes step 2.

It has now been fully appreciated that the above-mentioned drawbacks ofthis kind of part sequences can be minimised if the density of theliquid used in a fluidised bed step is lower than the density of theliquid used in the consecutive fluidised bed step.

The characterizing feature of the method is that the density of theliquid (liquid 2) used in step 2 is higher than the density of theliquid (liquid 1) used in step 1. The bed is kept in a fluidised stateduring the two steps. The fact that liquids of increasing densities areused for two consecutive steps means that the steps are carried out inthe same vessel with liquid 2 replacing liquid 1.

By the expression “the bed is kept in a fluidised state during the twosteps”πis meant that plug flow should essentially be maintained duringstep 1 and 2. This means that the plate number should be ≧5, preferably≧10 or ≧20 during substantially the whole period of time defined by thetwo steps. The plate number can be measured as described in theexperimental part of WO 9717132.

The density of a liquid used in a step is the density of the liquid asapplied to the fluidised bed, i.e. not including the density change thatmay occur during a step.

In addition to step 1 and step 2, one or more additional steps may bepresent in the actual sequence used. These extra steps may be selectedamong equilibration steps, capture steps, washing steps, releasingsteps, cleaning steps and regeneration steps and any other step that maybe available. One or more up to all such extra steps may be in fluidisedmode that preferably is carried out in the same vessel as the partsequence comprising steps 1 and 2. Steps that are not carried out influidised mode are supposed to be carried out in packed bed mode.Typical steps that are carried out in packed bed mode are releasingsteps and regeneration steps and equilibration steps and combinedregeneration/equilibration steps. Packed bed mode steps can be performedeither with upward or downward flow as is commonly known for this kindof beds. Step 1 may be consecutive to a fluidised bed step or to asequence of consecutive fluidised bed steps that together with steps 1and 2 form a sequence that utilizes liquids of increasing densities.Similarly step 2 may have an immediate subsequent fluidised bed step oran immediate subsequent sequence of fluidised bed steps that togetherwith steps 1 and 2 forms a sequence that utilizes liquids of increasingdensities.

The inventive concept is preferably applicable when at least one of step1 and step 2 is a functional step, for instance selected among a-fabove. Examples of part sequences in which both step 1 and step 2 arefunctional steps are:

Alternative Step 1 Step 2 I Washing step Releasing step II Releasingstep Cleaning step III Releasing step Releasing step with a first with asecond releasing agent releasing agent IV Capture step Washing step VWashing step with Washing step a first washing With a second liquidwashing liquid VI Density decreasing Any functional step step a-f VIICleaning step Regeneration step or equilibration step VIII Equilibrationstep Capture step

The table assumes that the liquids have been selected so that step 2 hasan increased density compared to step 1.

Density decreasing step: The feature of having fluidised bed steps inwhich a denser liquid is coming before a lighter liquid may haveadvantages when dealing with dense and viscous liquids, for instancecapture liquids. In these cases it may be difficult to increase thedensity further. This will be overcome by having a zone of lighterliquid (liquid 1, step 1), for instance a “wash solution”, to pass thebed, and then in the next step (step 2) increase the density of theliquid (liquid 2). The drawback is that the risk for bed turbulence willincrease but the liquid exiting the bed will anyhow be lighter than thedense liquid used prior to liquid 1. Liquid 2 can be, for instance atrue washing liquid, with an increased density relative the “wash”solution. With respect to the step that precedes step 1, this principlemay be applicable to any of steps a-f but in particular to step 1 beinga capture step.

The increase in density when going from step 1 to step 2 includes addingdensity-increasing agents to the liquid used in step 1 (“wash”solution). These agents should not decrease the binding of the compoundto the particles. Typical agents are uncharged soluble compounds such asuncharged compounds having carbohydrate structure. See below.

Step 2 may be used to keep the liquid used in the preceding step(step 1) and consecutive step (step 3) physically apart in the vesselused. In this variant steps 1 and 3 may be selected from steps a-fabove. By applying the principles of the invention, the liquid used instep 2 will have a density intermediate to the densities of step 1 andstep 3. This variant of the invention is particularly useful when step 1is a releasing step. During a releasing step the density of the liquidused will increase which in turn will cause a dilution of the sample andan increased volume. It may therefore be advantageous to elute with adenser liquid immediately after the releasing step, i.e. before thecleaning step or before a second compound is released, possibly byanother releasing agent and/or by a liquid having a higher densitycontaining the same releasing agent. The liquid used in step 2 of thisvariant may be-the same as the liquid used in step 1 but with adensifying substance added. This substance may be the same as ordifferent from the releasing agent in liquid 1. Other alternatives fordensifying agent are glycerol, other carbohydrates, salts etc.

A packed bed mode step may be inserted in the actual sequence used ifthere is a need to bring down the density of the liquid of a subsequentstep. For instance after a certain step, it may not be feasible orpractical to increase the density further. This use of packed mode bedsteps provides a simple and practical way of making a process accordingto the invention cyclic. The liquid used in this kind of packed bed modesteps is preferably less dense than the two liquids used in the closestsurrounding steps. Once the density of the liquid for a step has beenreduced then liquids of increasing densities can be used in consecutivesteps. In principle any step a-f above can be carried out in packed bedmode as described in this paragraph. For typical packed bed mode stepssee above.

An alternative way for enabling cyclic processes is to use part sequenceVI in the table above provided that the liquid in step 1 has asufficiently low density. This in practice means that a turbulent bedhas to be accepted in this step.

Great advantages will be accomplished in case step 1 is a washing or areleasing step.

An increase in density can be achieved by increasing the concentrationof a substance that is soluble in the liquid used and has adensity-increasing effect on the liquid. For aqueous liquids, typicalexamples of substances are salts such as halides (typically chlorides),phosphates, sulphates etc, for instance soluble metal and ammonium saltsthereof, and uncharged substances such as soluble carbohydrates, forinstance glycerol and other mono- or oligosaccharides. With respect toorganic compounds they should as a rule have a density higher than theliquid in order to provide an increased density when added to theliquid. Typically they should have a relatively large molecular weight,for instance reflected in number of carbon atoms being ≧3, such as incarbohydrates with ≧4 carbon atoms.

It is important to select the density-increasing agent so that it willnot interfere in an undesired way with the binding between the compoundand the particle in the step involved. In steps preceding a releasingstep the agent should not be able to act as a releasing agent for therelease intended in the step or in any other subsequent releasing stepsIn releasing steps the agent should not be able to counteract therelease intended.

The required relative difference in density between two consecutivefluidised bed steps will depend on various factors, for instance desirednumber of theoretical plates. This number in turn will depend on thecolumn design including the distributor design. Our results so farachieved suggest that the relative increase in density can be as low as1/10000 between two consecutive fluidised bed steps in case the systemis optimised to a plug flow corresponding to >35 theoretical plates inthe fluidised/expanded bed. Thus the relative increment in density foreach consecutive fluidised bed step can be ≧1/10000, such as ≧1/1000 or≧1/100 or ≧1/10 of the density of the liquid used in the immediatelypreceding fluidised bed step (for instance selected from steps a-f asdefined above). Depending on the system used, the number of theoreticalplates can be down to 5 provided that the relative density differencefor the liquids is sufficiently high between two consecutive fluidisedbed step. Thus systems providing, for instance, ≧5 such as ≧15 and ≧35theoretical plates in the fluidised bed may be used.

An increase in density is often accompanied by an increase in viscosity.Some substances have a more pronounced ability to increase the viscositythan others. This may have unfavourable effects in fluidised bedsystems. It may therefore be beneficial to switch from a pronounced to aless pronounced viscosity increasing substance when increasing thedensity of the fluidising liquid between two fluidised bed steps.

In absolute figures the density of the liquid used should be above thepure liquid without any density-increasing agent added. The upper limitis determined by the density of the particles and/or by practicalconsiderations, such as costs for density-increasing materials. Foraqueous liquids this means that the density of the liquids forconsecutive fluidised bed steps may change within the interval 0.98 to1.20 or to 1.50 g/cm³, with preference for 1.00 to 1.15 g/cm³. The lowerlimit 0.98 g/cm³ accounts for the fact that density-decreasing agentsmay be added, such as water-miscible organic solvents, for instancemethanol, ethanol etc. By the use of heavier particles, for instancewith densities ≧1.20 g/cm³, the upper limit of the density interval canpotentially be extended upwards and more dense liquids could accordinglybe used. This means that aqueous liquids used in two consecutivefluidised bed steps according to the invention may have a difference indensity in the range starting just above 0 and going up to at least 0.52g/cm³ with preference for at least 0.22 g/cm3. Analogous ranges can beset up in case one selects to use non-aqueous liquids.

The density of the particles should be ≧1.05 g/cm³, preferably ≧1.14g/cm³, and even ≧1.20 g/cm³ such as ≧1.30 g/cm³. An upper limit of 5-6g/cm³ can be envisaged. Suitable particles are described in WO 9218237(Amersham Pharmacia Biotech AB); WO 9717132 (Amersham Pharmacia BiotechAB); WO 9833572 (Amersham Pharmacia Biotech AB); and WO 9200799(Kem-En-Tek/Upfront Chromatography A/S). Suitable particles oftencontain inorganic material as a densifying material. Suitable particlesmay also contain synthetic polymers. Polymers can be divided into purelysynthetic polymers, semisynthetic polymers and biopolymers. Syntheticpolymers may have monomeric units selected amongst acryl amides,methacrylamides, hydroxy alkyl acrylates, hydroxy alkyl methacrylates,styrenes, divinyl benzenes etc. Semisynthetic polymers comprise forinstance cross-linked biopolymers and copolymerisates thereof andgrafted polymers exhibiting structures originating from biopolymers.Biopolymers comprise polysaccharides, such as dextran, agarose,cellulose, starch and pullulan. Well known particles that have been usedfor fluidised bed applications are sold under the trade mark Streamline(Amersham Pharmacia Biotech AB, Uppsala, Sweden) and belong to a groupof particles comprising both density increasing material, ofteninorganic, and hydrophilic organic material, typically polymeric.

The process temperature for various steps involved depends on the liquidused and the compound to be captured, among others. For aqueoussolutions the process temperature may be VIII Equilibration step Capturestep from 0° C. up to e.g. 70-90° C. although for practicalconsiderations the temperature is often in the interval 0-50° C. Forother liquids other ranges apply.

The inventive method has its largest use in processes of relativelylarge productivity. This means that the flow velocities used should beat least 70-3000 cm/h, preferably from 80-90 cm/h and upwards. Thevessels should have a cross sectional area that typically corresponds tothe area of a square having a side of at least 10 cm, such as at least15 cm. The cross-sectional area referred to is perpendicular to theliquid flow fluidising the particles.

As discussed above, the actual sequence may comprise one or more stepsthat are best performed in packed bed mode, possibly by reversing theflow relative the particles. By starting from a step in fluidised modein a traditional vessel, this may be accomplished by allowing theparticles to sediment to a “packed bed” and then apply an upward ordownward flow through the vessel. An alternative utilizes a tiltablevessel that is tilted 180° when changing bed mode. See FIGS. 7a-b of ourcopending International Patent Application deriving from SE 9803813-6and SE. This type of change in flow direction may be particularlyvaluable in case the particles are to be regenerated, for instance to beused in a second run of the same process. The advantages derives fromthe fact that the cleaning step often makes use of the liquid with thehighest density while a combined regeneration/equilibration steputilizes a liquid of low is density. An alternative to a packed bed modestep, for instance via tilting, may be to accept a lowered plate numberduring the releasing step and perform the step under fluidisingconditions with a liquid having a lowered density compared to thepreceding step. See above.

Packed bed steps may be combined with fluidised bed steps in devicesdesigned therefore. See copending International Patent Application withpriority from SE 9803813-6 and SE 9803737-7. Hence, the full actualsequence of the inventive process may be carried out in one commonvessel device in which the collector arrangement is maintained at afixed distance from the distributor arrangement during consecutivefluidised bed steps. The preferred type of vessels thus may have fixedlymounted collector and distributor arrangements. This does not excludethat the full sequence also can be performed in a vessel having amovable outlet adapter, for instance as described in WO 9520427(Amersham Pharmacia Biotech AB) and WO 9218237 (Amersham PharmaciaBiotech AB). Neither does it exclude a system of vessels in whichdifferent vessels are dedicated to fluidised bed steps and packed bedsteps, respectively (see FIGS. 8-10 in copending International PatentApplication deriving from SE 9803818-6 and SE 9803737-7).

The above-mentioned density ranges for liquids refer to densitiesmeasured at the actual process temperature. For particles the densitiesrefer to particles in the wet state having been soaked with the pureliquid used, for instance water. Plate numbers refer to those havingbeen obtained by the method described in WO 9717132.

Applications in Which the Invention can be Used

The invention is primarily used in liquid chromatography techniques.Examples are size exclusion (gel permeation) chromatography andadsorption techniques and techniques involving formation of covalentbonds between the particles and the compound to be removed from theliquid. Adsorption techniques are also called affinity chromatography.The important variants are ion exchange chromatography and techniquesbased on other affinity principles, such as bioaffinity, hydrophobicinteraction (HIC), chelating interaction etc. The structure on theparticles causing adsorption is often called affinity ligand or affinitystructure.

The compound to be captured on the particles may be ions, for instancemetal ions, and inorganic and organic compounds, for instancebiomolecules, such as proteins, carbohydrates, lipids, amino acids,hormones etc. In case of proteins they may have been producedrecombinantly in host cells (bacteria, yeast, mammalian, plant andinsect cells, for instance), by in vitro translation or in transgenicanimals, such as transgenic mammals and transgenic avians, for instancebudgerigars. In particular production of human proteins in cows, sheeps,goats, horses etc may be mentioned. Important proteins are native andrecombinant forms of plasma proteins, such as blood coagulation factors,immunoglobulins, ATIII, α1-antitrypsin, serum albumin etc; whey proteinssuch as lactoferrin and lactoperoxidase; enzymes; peptide or proteinhormones such as growth hormones, insulin etc; erythropoetin; proteinantigens and their fragments to be used, for instance, as vaccines oragents in hyposensitization therapy; and other proteins that are oftherapeutic interest. Among blood coagulation factors FVIII, FVII, FIXetc may be mentioned. Among immunoglobulins various forms of monoclonalantibodies (IgA, IgD, IgE, IgG, IgM) including fragments and fused formsthereof may be mentioned. Industrial enzymes such as those used inwashing powders and in other compositions intended for cleaning are ofpotential importance.

The samples to be applied to the fluidised bed are liquids containingthe compound to be bound to the particles in the capture step. Thisincludes fermentation broths, and other biological fluids derived fromanimals, such as mammals and other vertebrates, and evertebrates. Inparticular it includes transgenic animals as discussed above. Particularbiological fluids from animals are blood, serum, urine, milk (includingwhey) etc and other samples containing the biomolecules discussed abovetogether with sticky and/or particulate components.

The original sample may have undergone a number of pretreatment stepsbefore being applied in a capture step. Pretreatment steps may bedilution, concentration, desalting, removal of specific components,centrifugation, filtration, dialysis, ultrafiltration, pH-adjustmentsetc. A typical procedure is to dilute the sample in a buffer providingthe same conditions as the buffer used in the equilibration step. Analternative is to equilibrate the particles to the conditions providedby the sample. These procedures are typically carried out after theappropriate pretreatments of an original sample.

The invention will find uses within a large variety of technical fields,such as food industry, water purification and water deionisation, drugmanufacturing, metal refining etc,

A particular important aspect of using density differences as describedherein is for working up a compound from a sample deriving from abiological fluid of an animal, in particular a transgenic animal. Inthis aspect the process as such comprises an actual sequence of stepswith characteristic features as defined above. The biological fluidsconcerned and their origin have been discussed above. The biologicalfluid concerned are primarily those that contains particulate and/orsticky components and/or are more or less highly viscous, for instanceblood, serum, plasma, milk, whey etc. The compounds are the same asdiscussed above.

The preferred modes of the invention utilize vessels and systems asdescribed in copending International Patent Application derived from SE9803813-6 and SE 9803737-7.

The invention will now be illustrated in the experimental part. Theinvention is further defined by the appended claims.

EXPERIMENTAL PART Test of Using Density Differences in Preventing MixingBetween Subsequently Incoming Liquids in a Liquid Fluidised Bed

The background of this test is to be able to run the column in expandedmode throughout all the operating steps without loosing performance dueto instability of the bed (mixing, channeling etc.). The theory was thatthe density of the liquids is the key factor whether two differentliquids will mix or not in a fluidised bed and not the viscosity of theliquids. This means that a heavy liquid that is pumped into an expandedbed column (even distribution of liquid) which contains a lighterliquid, will create a sharp boundary between the two liquids and nomixing will occur. Whilst on the other hand, a light liquid pumped intoa heavy liquid will cause severe mixing. By using increasing densitiesfrom liquid to liquid no mixing will occur and thereby a minimum ofbuffer consumption will be gained.

Experiment

A conventional column (200 mm in diameter and 1000 mm in height) withthe existing distributor design was used (perforated plate with a mesh;Streamline, Amersham Pharmacia Biotech AB, Uppsala, Sweden). StreamlineDEAE gel was used in this experiment. Five different liquids were pumped(at 300 cm/h) into the column from bottom to top in the following order:

Liquid Density Comments 50 mM NaCl 1.000 The bed was equilibr. ½ hr 5%(dry weight) yeast 1.017 More viscous than 50 mM susp. NaCl 10.6%glycerol solution 1.022 More viscous than the yeast susp. 0.82M NaCl1.030 Less viscous than the glycerol sol. 1M NaOH 1.040 More viscousthan 0.82M NaCl

The result was sharp boundaries between the different liquids in thepresence of a fluidised bed and thereby no mixing of the liquids.

This experiment proved that the liquid density is the governing factorwhen it comes to stable non-mixing behaviour between different liquidseven in the presence of a fluidised bed.

What is claimed is:
 1. A liquid chromatographic process performed at least partly in a fluidised bed contained in a vessel and comprising particles fluidised by an upwardly directed liquid flow, said process comprising (a) capturing one or more compounds of a sample by the particles; and (b) washing and/or releasing the particles in a fluidised bed through which a liquid flow is passing, wherein a liquid (liquid 1) used in the wash or the releasing step (step 1) is immediately followed by adding a second liquid (liquid 2, step 2) having a higher density than liquid 1 while maintaining the bed in a fluidised state.
 2. The process of claim 1, wherein the liquid 2 (a) does not have any significant release effect or final cleaning effect; and (b) is used for separating the liquid 1 containing the released compound from a liquid 3 (step 3) that is added subsequent to liquid 2, and for instance is used for cleaning.
 3. The process of claim 2, wherein liquid 1 and liquid 2 have a density difference caused by a difference in concentration of a soluble substance.
 4. The process of claim 2, wherein the liquid 1 contains a releasing agent and is used for release of a compound captured on the particles and the liquid 2 has an increased density compared to liquid 1 due to the presence of a substance other than the releasing agent.
 5. The process of claim 1, wherein the vessel has a cross sectional area corresponding to the area of a square having a side of at least 10 cm.
 6. The process of claim 1, wherein (a) the vessel has an inlet end and an outlet end; and (b) the distance between the inlet end and the outlet end are kept essentially constant during at least two consecutive fluidised bed steps.
 7. The process of claim 1, wherein either (a) the distributor and collector arrangements of the vessel are fixedly mounted, or (b) the outlet end is movable relative the inlet end.
 8. A liquid chromatographic process performed at least partially in a fluidised bed contained in a vessel having an actual sequence of steps comprising (a) capturing a sample, derived from an animal, on the particles; and (b) two consecutive steps (step 1 and step 2) in which the bed is fluidised by an upward liquid flow of two different liquids (liquid 1 and liquid 2) passing through said vessel, wherein the liquid 2 has a density that is higher than the density of the liquid
 1. 9. The process of claim 8, wherein step 1 is a releasing step and step 2 is a cleaning step.
 10. The process according to claim 8, wherein step 1 is a washing step and step 2 is a releasing step.
 11. The process according to claim 8, wherein step 1 is an equilibration step and step 2 a capture step.
 12. The process according to claim 8, wherein step 1 is a density-decreasing step which is preceded by an additional step utilizing a liquid having a higher density than used in step
 1. 13. The process according to claim 12, wherein step 2 is a washing step.
 14. The process according to claim 8, further comprising a releasing step which is performed in packed bed mode.
 15. The process according to claim 8, wherein the capture step is in fluidised bed mode.
 16. The process according to claim 8, wherein the process is cyclic with each cycle ending with a regeneration step that is the equilibration step for a subsequent cycle.
 17. The process according to claim 15, wherein the release step, or the cleaning step or the regeneration/equilibration step is performed in packed bed mode.
 18. A liquid chromatographic process carried out in a vessel and having an actual sequence of steps comprising (a) at least a capture step in which a sample containing one or more compounds which are derived from cultured mammalian cells are bound to the particles and (b) two consecutive steps (step 1 and step 2) in which the bed is fluidised by an upward liquid flow passing through said vessel, wherein a liquid used in step 2 (liquid 2) has a density that is higher than the density of a liquid used in step 1 (liquid 1). 